Journal:

Article Title:

doi:

Figure Lengend Snippet: The WNT inhibitor IWR1 effectively enhances the differentiation efficiency of pPGCLCs. (A) Fluorescence microscope images and flow cytometry of NT reporter transgenic male pgEpiSCs cultured for 8 days with induction of SOX17 in the presence of cytokines: control (no cytokines), BESLY, BESLY + CHIR, BESLY + IWR1, BESLY + XAV939, BESLY + IWP2. Scale bars: 100 µm. (B) Bar graph quantification of the percentage of NANOS3‐tdTomato‐positive cells in pgEpiSCs (control, no cytokines) and Day 8 aggregates under the indicated treatments. Statistical significance was determined by t ‐test: ns, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars represent mean ± SEM. Biologically independent experiments, n = 3. (C) RT–qPCR analysis of gene expression in NT‐positive cells derived from male pgEpiSCs induced for 2, 4, 6, and 8 days with SOX17 transgenes in the presence of cytokines (BESLY) with or without IWR1. Values are normalized to GAPDH and presented as mean ± SD relative to untreated day 0 controls. Biologically independent experiments, n = 3. (D) IF analyses of the expression of SOX17, OCT4, NANOG, and DDX4 in NT + (red) cells of aggregates induced from transgenic male pgEpiSCs after 8 days induction of SOX17 in the presence of cytokines (BESLY + IWR1). The cells were counterstained with DAPI (gray), and merged images are shown on the right. Scale bars: 50 µm. (E) Principal component analysis (PCA) of global gene expression profiles during PGCLC differentiation. Cells were treated with IWR1 (+, triangles) or untreated (−, circles) at indicated time points (days 2, 4, 6, and 8). Dashed and solid lines indicate the differentiation trajectories under control (BESLY) and IWR1‐treated (BESLY + IWR1) conditions, respectively. (F) Heatmap of lineage‐specific gene expression during pPGCLC differentiation. Genes are grouped by functional lineage (WNT, mesoderm, PSC, PGC). Samples are ordered by time point (2d–8d) and treatment condition (IWR1−/+). Color scale represents relative expression levels. (G) Scatter plots comparing PGC gene expression vs. pluripotency gene expression in male (left) and female (right) PGCLCs relative to pgEpiSCs. Highlighted genes include key PGC markers and pluripotency markers. (H) Combined single‐cell and bulk RNA‐seq PCA, integrating PGCLC differentiation data with in vivo PGC. Clusters represent E11 ectoderm, E14 PGC, E31 PGC, male/female pgEpiSCs, and male/female PGCLCs.

Article Snippet: The 3i/LAF medium was prepared by adding 1 μ m CHIR99021 (Selleckchem, S1263), 2.5 μ m IWR1endo (Selleckchem, S7086), 1 μ m WH4023 (Selleckchem, S7565), 10 ng/mL recombinant human LIF (PeproTech, 300–05), 25 ng/mL Activin A (PeproTech, 120‐14E), and 10 ng/mL FGF2 (PeproTech, 100–18B).

Techniques: Fluorescence, Microscopy, Flow Cytometry, Transgenic Assay, Cell Culture, Control, Quantitative RT-PCR, Analysis, Gene Expression, Derivative Assay, Expressing, Functional Assay, Single Cell, RNA Sequencing, In Vivo

Journal:

Article Title:

doi:

Figure Lengend Snippet: Cross‐species comparison of PGCLC derivation and expansion characteristics.

Article Snippet: The 3i/LAF medium was prepared by adding 1 μ m CHIR99021 (Selleckchem, S1263), 2.5 μ m IWR1endo (Selleckchem, S7086), 1 μ m WH4023 (Selleckchem, S7565), 10 ng/mL recombinant human LIF (PeproTech, 300–05), 25 ng/mL Activin A (PeproTech, 120‐14E), and 10 ng/mL FGF2 (PeproTech, 100–18B).

Techniques: Comparison, Inhibition